Respiratory syncytial virus (RSV) is the most important cause of respiratory infection in infants and is a significant cause of disease in immunocompromised patients and the elderly. In addition, RSV is closely related to other highly pathogenic viruses, such as Ebola and Nipah, and is a good model for studying their molecular biology. RSV has an RNA genome that is transcribed and replicated by the virus polymerase to produce mRNA and progeny genomes. The focus of this project is the characterization of the interaction of the RSV polymerase complex with its promoter in the RSV genome. This will be achieved by addressing three major aims. (1) The region of the RSV polymerase that interacts with its binding site in the promoter will be identified using surface residue modification and UV cross-linking techniques. Identification of the interacting site will be confirmed by reverse-genetics, using a minigenome assay that allows analysis of RSV transcription and replication in an intracellular environment. (2) The mechanism of encapsidation initiation, necessary for the elongation stage of RNA replication will be examined. Mutations will be made in the encapsidation signal in the full-length clone of RSV and a forward-genetics approach will be applied to identify which virus protein, and which region within that protein, it might interact with. (3) Virus and cellular proteins that interact with the polymerase complex will be identified using immunoprecipitation of polymerase complexes, mass spectrometry, and Western blotting. The roles of associated cellular proteins during different stages of RSV RNA synthesis will be determined by inhibiting their expression and examining RSV RNA synthesis in the minigenome system and RSV infected cells. The effects of promoter mutations that are known to differentially modulate transcription and replication will be investigated to determine if they affect the composition of the polymerase complex that binds the promoter. This research will provide information regarding the composition and regulation of a protein-RNA complex that plays a key role in RSV multiplication. In the long-term, this work will allow detailed structural analysis of this complex to be performed, which could aid in design of inhibitors to control RSV infection, and could identify mutations that could be made in the virus genome to generate a live- attenuated RSV vaccine. PUBLIC HEALTH RELEVANCE: Respiratory syncytial virus (RSV) is a major human pathogen, responsible for significant numbers of pediatric hospitalizations and for seasonal disease in the elderly. The aim of this project is to progress our molecular understanding of a critical step in the virus multiplication cycle, with the long-term goal of developing antiviral drugs and vaccines to control RSV disease.